The overall goal of this research project is to determine the contribution of arsenic (As) exposure to human disease risk. There is growing worldwide concern about the human health effects of chronic, low level arsenic exposure. Arsenic in drinking water has been associated with an increased risk of developing type 2 diabetes, vascular and cardiovascular diseases, reproductive and developmental problems, and several kinds of cancer, notably lung, skin, bladder and liver cancer. We had previously shown that arsenic inhibited glucocorticoid hormone-mediated transcriptional gene regulation. We hypothesize that direct biochemical disruption of OR function by arsenic contributes to the pathophysiology of the diseases associated with chronic arsenic exposure. Recent experiments demonstrated that mutant GRs lacking either the N-terminal domain or the C-terminal ligand-binding domain had a similar response to As as wild-type OR. This suggested that the effects of arsenic are primarily mediated through the middle DNA binding domain (DBD). We also have preliminary data suggesting that As binds stoichiometrically to GR at very low intracellular concentrations of As. The specific goal of this project is to determine the biochemical basis for effects of arsenic on GR signaling, focusing principally on As effects on the DBD of OR. In particular, using model mammalian hepatocyte-derived cell lines, wild-type and mutant forms of OR, genetic constructs containing model GR-responsive genes, and various biochemical and genetic techniques, we will examine this question in detail with the following specific aims: 1) Determine the arsenic-OR binding stoichiometry and site(s) of interaction using mass spectrometry and site-directed mutagenesis of OR; 2) Determine the effects of arsenic on the normal functions of the GR DBD, examining in particular whether As alters: a) the formation of cytosolic GR dimers or their transport to the nucleus using mutant and tagged GRs in combination with immunoprecipitation and Western analysis; b) OR monomer-timer interactions with their glucocorticoid response element (GRE) DNA recognition sequences using gel shift and BlAcore analyses; or c) the interaction of OR-GRE complexes with co-activators and other transcription factors using a Chromatin Immuno-Precipitation (ChIP) assay; and 3) mutational analysis of the OR DBD to confirm the key results of specific aims 1 and 2. The goal of these aims is to develop a more detailed understanding of the molecular basis for the effects of arsenic on steroid receptor signaling. This will be important for determining the contribution of these effects to the overall human health effects of arsenic.